Screening for alpha1-antitrypsin deficiency in Lithuanian patients with COPD

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1 Respiratory Medicine (28) 12, 165e1658 available at journal homepage: SHORT COMMUNICATION Screening for alpha1-antitrypsin deficiency in Lithuanian patients with COPD Brigita Sitkauskiene a, Danielius Serapinas a, Ignacio Blanco b, Enrique Fernández-Bustillo c, Sabina Janciauskiene d, Raimundas Sakalauskas a, * a Department of Pulmonology and Immunology, Kaunas University of Medicine, Eiveniu 2, LT-59, Kaunas, Lithuania b Internal Medicine Division, Respiratory Diseases Branch, Hospital Valle del Nalon, Langreo, Principado de Asturias, Spain c Bio-statistics Unit, Hospital Central de Asturias, Oviedo, Principado de Asturias, Spain d Department of Clinical Sciences, University Hospital Malmö, Lund University, Sweden Received 7 January 28; accepted 2 July 28 Available online 21 August 28 KEYWORDS Alpha1-antitrypsin deficiency; Chronic obstructive pulmonary disease; Screening; Population studies Summary Background: Alpha1-antitrypsin (AAT) deficiency is an under-diagnosed condition in patients with chronic obstructive pulmonary disease (COPD). The objective of the present screening was to estimate the AAT gene frequency and prevalence and to identify AAT deficiency cases in a large cohort of Lithuanian patients with COPD. Methods: A nationwide program of AAT deficiency was conducted in 1167 COPD patients, defined according to the GOLD criteria. Patients were collected from outpatient clinics in five different Lithuanian regions (Kaunas, Vilnius, Siauliai, Klaipeda and Alytus). AAT serum concentrations were measured by nephelometry; PI-phenotypes characterized by isoelectricfocusing. Results: Mean age and FEV 1 were 62. (1.3) and 5.7% (1.9), respectively. Ninety-one AAT deficiency genotypes ( MZ, 39 MS, 1 SS, 3 SZ and 8 ZZ) were identified. Calculated PI*S and PI)Z frequencies, expressed in per 1, were 18.8 (95% CI: 13.9e25) and 25.3 (95% CI:.e32.7), respectively. The calculated AAT gene prevalence (HardyeWeinberg principle) was: 1/1.9 for MM, 1/28 for MS, 1/281 for SS, 1/2 for MZ, 1/19 for SZ and 1/1565 for ZZ. Calculated Odds ratio (OR) for PI)Z in COPD vs. Lithuanian healthy people was of 1.87 (P Z.). Conclusion: The OR for each genotypic class demonstrated a significant increase of MZ, SZ and ZZ genotypes in COPD patients. The results of the present study, with a significant number of * Corresponding author. Tel.: þ ; fax: þ address: raimundas.sakalauskas@kmuk.lt (R. Sakalauskas) /$ - see front matter ª 28 Elsevier Ltd. All rights reserved. doi:1.116/j.rmed

2 AAT deficiency screening in COPD in Lithuania 1655 ZZ individuals detected, support the general concept of targeted screening for AAT deficiency in countries like Lithuania, with a large population of COPD patients and low awareness among care-givers about this genetic condition. ª 28 Elsevier Ltd. All rights reserved. Introduction Chronic obstructive pulmonary disease (COPD) is a prevalent and costly disease characterized by progressive airflow limitation. It arises as an abnormal inflammatory response of the lung to long-term tobacco smoking or toxic gas inhalation. The lung inflammation is further exacerbated by oxidative stress and proteolytic damage by proteinases. 1 The prevalence of COPD is appreciably higher in men over years of age who are current or former heavy smokers. However, there is a consistent evidence that only 15e3% of smokers develop COPD, and that some non-smokers may also develop chronic airflow obstruction, suggesting that the risk for COPD results from a gene-environment interaction. The best described genetic risk factor for COPD is severe hereditary deficiency of alpha1-antitrypsin (AAT), 2 a genetic disorder most commonly seen in Caucasians. 3 AAT is a circulating serine proteinase inhibitor secreted by the liver, which permeates most body tissues where it acts as an inhibitor of a range of proteolytic enzymes. About 1 genetic variants of AAT have been identified, but most of the pathologies are related to AAT deficiency linked to the Z allele: in clinical practice, 96% of AAT deficiency patients have a ZZ genotype. The remaining % belong mostly to SZ, and other rare deficiency (i.e., Mmalton and Siiyama) or null genotypes. 2 AAT deficiency is an under-diagnosed condition worldwide. Recent guidelines from both the World Health Organization and the American Thoracic Society/European Respiratory Society recommend the establishment of screening programs for the detection of AAT deficiency in patients with COPD, since the detection of coincident AAT deficiency could lead to family screening, appropriate management (including lifestyle changes such as quitting smoking and replacement therapy in selected cases), and specific counselling for these patients and families. 2, Therefore, the main objective of this study was to identify the S and Z gene frequency in a cohort of 1167 COPD patients from five main cities of Lithuania. Methods Sample sources and subjects selection The study design was approved by the Regional Ethics Committee of the Kaunas University of Medicine. During the 2 months from January 25 to January 27, 158 patients with COPD were offered to participate in the study through referral by 22 physicians from the different Lithuanian regions (namely Kaunas, Vilnius, Siauliai, Klaipeda and Alytus). A total of 1167 COPD patients, who gave their informed consent, underwent further examination. Approximately 7% of the patients studied were previously diagnosed in outpatient medical clinics by primary care-givers and referred to the referral hospital to confirm diagnosis and start or review a proper management regimen. The remaining 3% patients were identified in hospital outpatient medical clinics by pulmonologists during routine ambulatory follow-up visits or when they were clinically stabilised after hospital discharge. Only patients who met the GOLD 1 spirometric criteria for COPD: (1) ratio of post-bronchodilator forced expiratory volume in 1 s (FEV 1 ) to forced vital capacity (FVC) less than.7, and (2) FEV 1 less than 8% of the predicted value were included in the study. After an appropriate physical examination, data on the symptoms of the patient and the diagnosis of COPD were also collected. Smoking history was calculated in pack-years as the product of tobacco use (in years) and the average number of cigarettes smoked per day/2 (years cig. per day/2). Sample collection and evaluation Blood samples were drawn in serum tubes, clotted at room temperature for 3e6 min and centrifuged for 15 min at rpm. Then, serum samples were immediately frozen at 7 C for further assay. The serum concentrations of AAT were determined by means of nephelometry using commercially available kits (Dade Behring Marburg GmbH, Germany) according to the manufacturer s instructions. The presence of PI)Z allele was checked by enzyme-linked immunosorbent assay (ELISA) kits (Euro-Diagnostica/Wieslab, Sweden), 5 qualitative method according to prepared standard guidelines for the product. AAT phenotyping was carried out by means of isoelectric-focusing (LKB Multiphor II and LKB Macrodrive 5 Constant Power Supply, Amersham Pharmacia Biotech, Piscataway, NJ, USA), as previously described. 6 Calculations and statistical analyses The methodology used in the present study for AAT gene frequency and prevalence calculations was the same as in previous publications. 3, Statistical comparisons were also done with data from two previous studies on AAT genetic epidemiology carried out on a total of 291 Lithuanian healthy adults. 7,8 Data for the Lithuanian population were obtained from Statistics Lithuania. 9 Descriptive statistics were used to tabulate the primary cohort database. Quantitative variables were expressed as means with standard deviations (SD). The values of serum AAT concentration were compared using Student s t-test. Some data were analyzed using the KruskaleWallis test and the ManneWhitney U-test. The Statistical Package for the Social Sciences software program was used to perform statistics analysis [SPSS Ibérica, Madrid, Spain]. The value of P <.5 was accepted for statistical significance.

3 1656 B. Sitkauskiene et al. Table 1 Demographic characteristics, AAT genotypes, spirometric values and AAT serum concentrations from a Lithuanian cohort of 1167 COPD patients AAT genotypes N Sex %M Age (SD) Cumulative pack-years (SD) FEV 1 /FVC (SD) FEV 1 % pred. AAT serum concentration MM (1.) 22. (12.) 5.8 (1.5) 6 (15.8) 16.7 (39.8) MS (7.8) 2. (11.6) 55.7 (11.2) 5.8 (16.1) 12 (11) MZ (11.2) 21.3 (12) 58.6 (9.5) 51.5 (1.5) 99 (1) SS ( ) SZ (2.8) 18 (2.8) 56.5 (6.3) 7 (.2) 77(.7) ZZ (11.3) (11) 36.2 (18.6) 38.5 (18.1) 3 (12.) Total (1.3) 22.1 (12.2) 5.7 (1.9) 6.5 (15.9) 158 (3.6) P-value Between all genotypic classes ZZ vs. remaining groups Data are presented as mean or %, unless otherwise stated. N: number; P: statistical significance level (<.5); AAT: alpha1-antitrypsin; %: percent; SD: standard deviation; M: males; FEV 1 : forced expiratory volume in one second expressed in percent of the predicted normal value. AAT serum concentrations measured by nephelometry, values expressed in mg/dl. Results As shown in Table 1, the cohort consisted of 1167 patients (82% males and 18% females) with moderate to severe COPD. The distribution of genotypes (number and percentage) was: 176 (92.2%) MM, (3.%) MZ, 39 (3.3%) MS, 1 (.1%) SS, 3 (.3%) SZ and 8 (.7%) ZZ. The mean age of the cohort was 62 years (1.3). The ZZ group compared to all the remaining groups was younger (5 years) but not significantly (P Z.6). Nearly 83% of the patients was current (57.6%) or former (25.2%) smokers of pack-years and 17.2% never smoked. Smoking status (pack-years) did not significantly differ between the current and former smoker groups. The mean value of FEV 1 was 6.5 (15.9), and the mean value of the ratio FEV 1 /FVC was 5.7 (1.9). There was no significant difference among all groups in FEV 1. The ZZ group showed a lower FEV 1 /FVC (36., P Z.1) compared to other genotype groups. The mean AAT serum concentration (mg/dl) was of 158 (3.6). As expected, we found significant differences in AAT serum concentrations between groups (P Z.), although there was an overlap among MS, MZ and SS AAT concentrations. The ZZ group showed a significantly lower AAT serum concentration (3 mg/dl, P Z.) relative to the other genotype groups. Calculated gene frequencies for PI)S and PI)Z were 18.8 (13.9e25) and 25.3 (.e32.7) per 1 (Table 2). Calculated prevalence of different AAT genotypes was: 1/1.9 for MM, 1/28 for MS, 1/281 for SS, 1/2 for MZ, 1/19 for SZ and 1/1565 for ZZ. Two previous studies of AAT genetic epidemiology in Lithuanian healthy adults showed a mean gene frequency (expressed in per 1) of 15.6 (95% IC: 12e2) for S and 13.6 (95% IC: 11e) for Z. 7,8 The odds ratio (OR) for PI)Z between COPD and healthy Lithuanian adults was 1.87 (1.3e2.7), P Z.. Discussion In the current study, we show for the first time that the PI)Z gene frequency is about two-fold higher among COPD patients compared to the general population of Lithuania. 7,8 The odds ratios for each genetic group among COPD patients were lower in the normal MM genotype, as predicted in the MS and SS deficiency genotypes, but significantly higher in the MZ, SZ, and ZZ deficiency genotypes. The most striking finding of the COPD cohort screening was the high number of ZZ genotypes. The ZZ patients showed very low serum concentrations of AAT, poorer spirometric values, and they were younger than the remaining COPD patients. Most of the studied COPD patients were men (82%). The prevalence of men among COPD patients in Lithuania could be explained by smoking habits. According to the Finbalt Health Monitor program, the number of men smokers in the general Lithuanian population is 6.9 times higher than the number of women smokers. 1 Table 2 Estimated PI * S and PI * Z gene frequency and genotype prevalence (MS, SS, MZ, SZ and ZZ) in 1167 COPD patients and joined cohorts of 291 healthy unrelated people (Refs. 7,8) from Lithuania Samples Gene frequency per 1 (95% CI) HardyeWeinberg equilibrium calculated prevalence (1/x) PI*S PI*Z MS SS MZ SZ ZZ Total COPD N: (13.9e25.) 25.3 (.e32.7) Healthy unrelated people N: (12.5e.6) 13.6 (1.7e17.) % CI: 95% confidence intervals. Calculated Odds Ratio for Z and S alleles in COPD vs. healthy general population were of 1.87 (1.3e 2.7), P Z. and 1.21(.82e1.78), P Z.32.

4 AAT deficiency screening in COPD in Lithuania 1657 ICELAND NORWAY FINLAND 13 RUSSIA DEMMARK SWEDEN 2 23 ESTONIA Aland Arch LATVIA Gotland BELARUS 11 8 RUSSIA LITHUANIA Z values are showed in grey circles Figure 1 Distribution of PI)S and PI)Z gene frequencies (expressed in 1) in Northern Europe countries. Overall, the results of our study show both similarities and differences to those reported in other European countries, where AAT deficiency detection programs have been carried out using different methodologies. For example, case control studies in other countries also showed an increase in the prevalence of PI)Z heterozygotes among patients with COPD compared with the control group (OR for the MZ phenotype: between 1.5 and 5). 11e1 The case-detection program of 2137 COPD patients in Spain revealed seven cases of ZZ deficiency (.37%). 11 In another study undertaken in Italy, the detection rate for ZZ was 6.%. 12 The higher rate of ZZ cases in the latter could be due to the design of the study, since only COPD patients with familial or clinically inferred AAT deficiency were selected. In contrast, another program carried out in Germany 13 with 16 patients showed no subjects with the ZZ phenotype. This is probably related to the sample composition which included individuals with different types of chronic respiratory diseases. In contrast, a recent study performed in Germany using a targeted screening program with 2272 subjects identified 335 patients with severe ZZ AAT deficiency, including 16 individuals with rare genotypes. 1 Our results closely parallel to the data received from a study carried out in Denmark where the frequency of AAT deficiency among COPD patients was found to be.8% for ZZ and 2.% for MZ genotype. 15 The highest incidence of the PI)S allele in Northern Europe (around 2e3 per 1) is found in the South of the Scandinavian Peninsula, followed by Latvia, and Denmark, and it significantly decreases through the North and the East. The Z deficiency allele is exceptionally high in Latvia relative to other countries worldwide, followed by southern Norway, Denmark, Southern Sweden and Estonia, and in decreasing frequency in Lithuania, Finland, Europe Russia and Iceland. 16 Previous studies performed by other investigators in the general population of Lithuania 7,8 reported a moderate prevalence of both S and Z alleles compared to other Northern Europe countries (Fig. 1). The prevalence of COPD in Lithuania has not been reported, but it is estimated to be similar to that in other European countries: 8e1% in the population > years. 17 This implies that w15, Lithuanian people may suffer from this disease, and that approximately 1 ZZ subjects remain undiagnosed in the COPD population. The results of the present study support the general concept of targeted screening for AAT deficiency in other Northern European countries with a high frequency of PI)Z and a large population of COPD patients. Conflict of interest The authors have no conflict of interest regarding this manuscript. Acknowledgments We acknowledge the expert collaboration of physicians: K. Malakauskas, K. Stravinskaite, J. Vebriene, V. Arbaciauskas, D. Jureleviciene, D. Susinskiene (Kaunas), D. Stakisaitis, D. Vaicius, R. Balciuviene (Vilnius), S. Naudziunas, L. Vasiljeviene, V. Globyte (Siauliai), A. Valavicius (Klaipeda), A. Kiziela (Alytus), for the enrolment of patients, V. Asmoniene, B. Tarutiene, and A. Babusyte for their

5 1658 B. Sitkauskiene et al. technical support. The authors also are indebted to Ms. J. Blanco (Licenciée en Traduction Univ. de Genève) for the editing of the final draft manuscript. This study was in part supported by the Scientific Foundation of the Kaunas University of Medicine (Project Grant PAR8), Swedish Medical research Council (SJ) and Baxter Company. References 1. Global Initiative for Chronic Obstructive Lung Disease (GOLD). Global strategy for the diagnosis, management, and prevention of COPD, executive Summary. 26. [accessed 8.7]. 2. American Thoracic Society/European Respiratory Society Statement: standards for the diagnosis and management of individuals with alpha1-antitrypsin deficiency. Am J Respir Crit Care Med 23;168:818e9. 3. Blanco I, de Serres FJ, Fernandez-Bustillo E, Lara B, Miravitlles M. Estimated numbers and prevalence of PI)S and PI)Z alleles of alpha1-antitrypsin deficiency in European countries. Eur Respir J 26;27:77e8.. De Serres FJ, Blanco I, Fernandez-Bustillo E. Estimating the risk for alpha-1 antitrypsin deficiency among COPD patients: evidence supporting targeted screening. COPD 26;3: 133e9. 5. Gershagen S, Janciauskiene S. ELISA for specific detection of PiZ-related alpha1-antitrypsin deficiency. Clin Chem 2;5: 27e1. 6. Pierce JA, Eradio BG. Improved identification of antitrypsin phenotypes through isoelectric focusing with dithioerythritol. J Lab Clin Med 79;9:826e Beckman L, Sikström C, Mikelsaar A, Krumina A, Kucinskas V, Beckman G. Alpha1-antitrypsin PI alleles as markers of West European influence in the Baltic Sea region. Hum Hered 99;9:52e5. 8. Stakisaitis D, Basys V, Benetis R. Does alpha-1 proteinase inhibitor play a protective role in coronary atherosclerosis? Med Sci Monit 21;7:71e Lithuania Statistics, [accessed 8.7]. 1. Grabauskas V, Klumbiene J, Petkeviciene J, Kinderyte G, Sackute A, Hellasoja V, et al. Health behaviour among Lithuanian adult population. Available from:. Publication of National Public Health Institute lithuania_finbaltreport22.pdf; 22. B12/ De la Roza C, Rodríguez-Frías F, Lara B, Vidal R, Jardí R, Miravitlles M. Results of a case-detection programme for alpha1-antitrypsin deficiency in COPD patients. Eur Respir J 25;26:616e Luisetti M, Massi G, Massobrio M, Guarraci P, Menchicchi FM, Beccaria M, et al. A national program for detection of alpha- 1 antitrypsin deficiency in Italy. Respir Med 99;93:169e Wencker M, Marx A, Konietzko N, Schaefer B, Campbell EJ. Screening for alpha Pi deficiency in patients with lung diseases. Eur Respir J 22;2:3e2. 1. Bals R, Koczulla R, Kotke V, Andress J, Blackert K, Vogelmeier C. Identification of individuals with alpha-1-antitrypsin deficiency by a targeted screening program. Respir Med 27;11:178e Dahl M, Tybjaerg-Hansen A, Lange P, Vestbo J, Nordestgaard BG. Change in lung function and morbidity from chronic obstructive pulmonary disease in alpha1-antitrypsin MZ heterozygotes: a longitudinal study of the general population. Ann Intern Med 22;136:27e De Serres F, Blanco I, Fernández-Bustillo E. PI)S and PI)Z Alpha-1 antitrypsin deficiency worldwide. A review of existing genetic epidemiological data. Monaldi Arch Chest Dis 27;67: 18e Lindberg A, Jonsson AC, Ronmark E, Lundgren R, Larsson LG, Lundback B. Prevalence of chronic obstructive pulmonary disease according to BTS, ERS, GOLD and ATS criteria in relation to doctor s diagnosis, symptoms, age, gender, and smoking habits. Respiration 25;72:71e9.